1
Plitidepsin has a positive therapeutic index in adult patients with COVID-19
requiring hospitalization
Authors: José F. Varona
1,2*
, Pedro Landete
3,4
, Jose A. Lopez-Martin
5
, Vicente Estrada
6,7
, Roger
Paredes
8,9
, Pablo Guisado-Vasco
10,11
, Lucía Fernández de Orueta
11,12
, Miguel Torralba
13,14
, Jesús
5
Fortún
15
, Roberto Vates
12
, José Barberán
1,2
, Bonaventura Clotet
8,9,16,17
, Julio Ancochea
3,4,18
,
Daniel Carnevali
10,11
, Noemí Cabello
19
, Lourdes Porras
20
, Paloma Gijón
21
, Alfonso Monereo
12
,
Daniel Abad
11,12
, Sonia Zúñiga
22
, Isabel Sola
22
, Jordi Rodon
23
, Nuria Izquierdo-Useros
24,25
,
Salvador Fudio
26
, María José Pontes
27
, Beatriz de Rivas
27
, Patricia Girón de Velasco
5
, Belén
Sopesén
5,28,29
, Antonio Nieto
30
, Javier Gómez
30
, Pablo Avilés
31
, Rubin Lubomirov
26
, Kris M.
10
White
32,33
, Romel Rosales
32,33
, Soner Yildiz
32,33
, Ann-Kathrin Reuschl
34
, Lucy G. Thorne
34
,
Clare Jolly
34
, Greg J. Towers
34
, Lorena Zuliani-Alvarez
35,36,37,38
, Mehdi Bouhaddou
35,36,37,38
,
Kirsten Obernier
35,36,37,38
, Luis Enjuanes
22
, Jose M. Fernández-Sousa
39
, Plitidepsin – COVID - 19
Study Group
‡
, Nevan J. Krogan
32,35,36,37,38*
, José M. Jimeno
5†
, Adolfo García-Sastre
32,33,40,41†*
.
Affiliations:
15
1
Departamento de Medicina Interna, Hospital Universitario HM Monteprincipe, HM
Hospitales, Madrid, Spain.
2
Facultad de Medicina, Universidad San Pablo-CEU, Madrid, Spain.
3
Hospital Universitario La Princesa, Madrid, Spain.
4
Universidad Autónoma de Madrid, Madrid, Spain.
20
5
Virology & Inflammation Unit, PharmaMar, S.A., Colmenar Viejo, Madrid, Spain.
6
Hospital Clínico San Carlos, Madrid, Spain.
7
Universidad Complutense de Madrid, Madrid, Spain.
8
Infectious Diseases Department, irsiCaixa AIDS Research Institute, Badalona, Barcelona,
Spain.
25
9
Hospital Germans Trias I Pujol, Badalona, Barcelona, Spain.
10
Internal Medicine Department. Hospital Universitario Quironsalud Madrid, Madrid, Spain.
11
Universidad Europea, Madrid, Spain.
12
Internal Medicine Department, Hospital Universitario de Getafe, Madrid, Spain.
13
Health Sciences Faculty, University of Alcalá, Madrid, Spain.
30
14
Guadalajara University Hospital, Guadalajara, Spain.
15
Hospital Universitario Ramón y Cajal, Madrid, Spain.
16
Universitat Autònoma de Barcelona, Barcelona, Spain.
17
Universitat de Vic, Universitat Central de Catalunya Barcelona, Spain.
18
Centro de Investigación en Red de Enfermedades Respiratorias (CIBERES), Instituto de
35
Salud Carlos III (ISCIII), Madrid
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprintthis version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.25.21257505doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
2
19
Infectious Diseases Department, Clinico San Carlos University Hospital, Madrid, Spain.
20
Internal Medicine, Hospital General de Ciudad Real, Ciudad Real, Spain.
21
Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario
Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
22
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-
5
CSIC), Madrid, Spain.
23
IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la UAB,
Bellaterra, Spain
24
IrsiCaixa AIDS Research Institute, Badalona, Barcelona, Spain.
25
Germans Trias i Pujol Research Institute (IGTP), Badalona, Barcelone, Spain.
10
25
Clinical Pharmacology Unit, PharmaMar, Colmenar Viejo, Madrid, Spain.
27
Medical Affairs, PharmaMar, Colmenar Viejo, Madrid, Spain.
28
Sylentis, S.A.U., Tres Cantos, Madrid, Spain.
29
Biocross, S.L., Valladolid, Spain
30
Bio Statistics Unit, PharmaMar, Colmenar Viejo, Madrid, Spain.
15
31
Preclinical Unit, Pharmamar, Colmenar Viejo, Madrid, Spain.
32
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY,
USA.
33
Global Health Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai,
New York, NY, USA.
20
34
Division of Infection and Immunity, University College London, London WC1E 6BT,
United Kingdom.
35
Quantitative Biosciences Institute (QBI), University of California San Francisco, San
Francisco, CA 94158, USA.
36
J. David Gladstone Institutes, San Francisco, CA 94158, USA.
25
37
Quantitative Biosciences Institute (QBI), Coronavirus Research Group (QCRG), San
Francisco, CA 94158, USA.
38
Department of Cellular and Molecular Pharmacology, University of California, San
Francisco, CA 94518, USA.
39
Pharmamar, Colmenar Viejo, Madrid, Spain.
30
40
Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at
Mount Sinai, New York, NY, USA.
41
Tish Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
* Corresponding author. Email: jfvarona@hmhospitales.com (J.F.V.); Adolfo.Garcia-
Sastre@mssm.edu (A.G-S.); Nevan.Krogan@ucsf.edu, (N.J.K.)
35
†
These authors contributed equally to this work.
‡ A full list of the authors and affiliations included in the Supplementary Materials.
All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprintthis version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.25.21257505doi: medRxiv preprint
3
Abstract: Plitidepsin is a marine-derived cyclic-peptide that inhibits SARS-CoV-2 replication at
low nanomolar concentrations by the targeting of host protein eEF1A (eukaryotic translation-
elongation-factor-1A). We evaluated a model of intervention with plitidepsin in hospitalized
COVID-19 adult patients where three doses were assessed (1.5, 2 and 2.5 mg/day for 3 days, as a
90-minute intravenous infusion) in 45 patients (15 per dose-cohort). Treatment was well
5
tolerated, with only two Grade 3 treatment-related adverse events observed (hypersensitivity and
diarrhea). The discharge rates by Days 8 and 15 were 56.8% and 81.8%, respectively, with data
sustaining dose-effect. A mean 4.2 log10 viral load reduction was attained by Day 15.
Improvement in inflammation markers was also noted in a seemingly dose-dependent manner.
These results suggest that plitidepsin impacts the outcome of patients with COVID-19.
10
One-Sentence Summary: Plitidepsin, an inhibitor of SARS-Cov-2 in vitro, is safe and
positively influences the outcome of patients hospitalized with COVID-19.
15
Key Words: COVID-19 treatment, plitidepsin, eEF1A, SARS-CoV-2
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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4
Introduction
As of April 2021, there have been approximately 135 million confirmed cases of COVID-19
reported to the World Health Organization (WHO), including 3 million deaths. (1) The lack of
effective antiviral therapies represents a glaring unmet need, not only for the treatment of the
current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, (2) but also
5
for potential future pandemics, which may originate from other emergent coronaviruses. (3-5)
Viruses, particularly single-stranded positive-sense RNA viruses, are ubiquitous in the sea,
where they participate directly or indirectly in the population dynamics of marine organisms. (6)
Thus, the sea may prove to be a reservoir of potent, naturally selected antiviral compounds.
One such compound, plitidepsin, is a cyclic depsipeptide originally isolated from a
10
Mediterranean marine tunicate (Aplidium albicans) and is structurally related to didemnins, some
of which (i.e., those isolated from Trididemnum solidum) have shown antiviral properties. (7, 8)
Plitidepsin targets eukaryotic translation elongation factor 1A (eEF1A) (9), which is one of the
most abundant protein synthesis factors in the eukaryotic cell, (9, 10) and a protein known to be
used by viruses to facilitate their replication inside their host. (11) The SARS-CoV-2
15
nucleocapsid (N) protein is a key element involved in packaging of the viral RNA genome, (12,
13) and interacts with eEF1A. This interaction might be essential for viral replication, as eEF1A
knockdown results in a significant reduction in virus replication. (12, 13)
Originally developed as a cancer treatment, plitidepsin has undergone an extensive clinical
development program. Specifically, several phase I and II clinical trials have been conducted to
20
explore different intravenous dosing schedules and infusion times (14-18). Pharmacokinetic and
safety properties of plitidepsin were gathered from these studies. Based on the results obtained
from a phase III clinical trial (ADMYRE) (19), the Australian Therapeutic Goods Administration
(TGA) approved the combination of plitidepsin with dexamethasone for the treatment of patients
with relapsed/refractory multiple myeloma in 2018. (20)
25
In this study, we describe the results from a Proof-of Concept clinical trial that explores the
potential of plitidepsin as a therapy for patients hospitalized with COVID-19.
Results
We have evaluated the antiviral activity of plitidepsin against different coronavirus species,
30
strains and variants (Materials and Methods). Treatment of Huh-7 cells with as little as 0.5nM of
plitidepsin inhibited infection of a human coronavirus 229E expressing GFP (Figure 1A). A
10
4
- fold decrease in SARS-CoV genomic RNA (gRNA) accumulation and a 10
3
-fold decrease
in virus SARS-CoV titers were observed in VeroE6 cells treated with 50nM plitidepsin. (Figure
S1 and Table S1). By comparison, and consistent with previous results (10, 21) plitidepsin
35
showed nanomolar efficacy against SARS-CoV-2-induced cytopathic effects on Vero E6 cells
with a half-maximal inhibitory concentration (IC
50
) of 0.038µM, at concentrations where no
cytotoxic effects were observed (CC
50
2.9µM) (Figure 1B). Moreover, plitidpesin maintained its
nanomolar potency against replication of early as well as recently emerging SARS-CoV-2
lineages, such as B.1.1.7, in human lung and gastrointestinal cell lines (Figure 1C). Noteworthy,
40
plitidepsin was more effective against both variants than remdesivir (Figure 1C).
To identify target human plasma concentrations of plitidepsin for SARS-CoV-2 infection we
developed an extrapolation from in vitro results, in line with current recommendations. (22) This
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5
approach integrated results from non-clinical studies described under Material and Methods
(Supplement), including in vitro drug sensitivity data for SARS-CoV-2 in Vero cells, human
plasma protein binding data (98%) (Table S2), and in vivo tissue distribution data in rats (lung-
to-plasma partition coefficient ratio of 543-fold (Table S3).
The target plasma and lung concentrations for plitidepsin were initially based on in vitro data
5
obtained by Boryung Pharmaceuticals, stablishing IC
50
of 3.26 nM and IC
90
of 9.38 nM
(Material & Methods). A validated pharmacokinetic population model of plitidepsin (23) was
used to simulate plasma exposures at different dose levels and infusion durations, so that
plitidepsin plasma profiles would reach 0.33 µg/L, and 0.96 µg/L, assuring target concentrations
in lung above the aforementioned in vitro IC
50
and IC
90
. A 3-day daily schedule was initially
10
selected to achieve sustained active exposures, under the hypothesis that an acute reduction of
the viral load would prevent the onset of the more severe inflammatory phase of COVID-19. The
predicted plasma concentrations of plitidepsin, at a dose of 1.5 mg infused intravenously over
90 min, were above the target IC
50
for the full treatment period and above the IC
90
for half of the
treatment period. The respective predictions after a dose of 2.5 mg were above the IC
90
during
15
most of the treatment period (Figure 2). Thus, we anticipated that the proposed range of doses
would result in stable active concentrations in critical anatomical compartments, such as lung,
for more than 120 hours. This model was later supported by White et al, who reported an IC
90
of
0.88 nM (10). To reach this target concentration in lung tissue, according to the above reasoning,
plitidepsin plasma concentration should be above 0.18 µg/L.
20
We subsequently designed the APLICOV-PC study [APL-D-002-20; EudraCT #2020-001993-
31; NCT #04382066] as a proof-of-concept clinical trial, exploring three dose levels of
plitidepsin (1.5 mg/day, 2.0 mg/day, and 2.5 mg/day, flat doses) for 3 consecutive days, as a 90-
min intravenous (IV) infusion, in adult patients with COVID-19 who required hospitalization.
The primary endpoints were related to safety, but secondary efficacy and pharmacodynamic
25
endpoints were also included (Material and Methods).
Enrollment began on May 12, 2020 and the study was completed by November 26, 2020. Here,
we present the data analyses by the cut-off date of December 10, 2020.
In total, 46 hospitalized COVID-19 patients were enrolled across 10 sites in Spain. Baseline
demographic and clinical characteristics are summarized in Table 1. One patient withdrew
30
consent before initiating study-specific procedures. The 45 patients who received treatment were
sequentially allocated to one of the three dose cohorts; whenever more than 1 dose cohort was
open, a patient was assigned by central randomization (Material and Methods).
Forty-four patients completed the study through day 31, with one patient in the 1.5 mg cohort
withdrawing from the study after the first infusion due to a grade 3 hypersensitivity reaction. The
35
average patient age was 52 years (range 31–84 years). Most patients were male (66.7%) and 80%
had co-morbidities (46.7% had two or more). The most commonly reported comorbidities were
obesity (22.2%), hypertension (20%), and type 2 diabetes mellitus (17.8%). The distribution of
comorbidities was similar between the three treatment cohorts.
Most patients had moderate COVID-19 (51.1%), according to FDA categorization (24), with
40
13.3% and 35.6% having mild and severe disease, respectively. Baseline chest X-rays showed
evidence of lower respiratory infection (infiltrates, unilateral pneumonia, or bilateral pneumonia)
in 41 of 45 treated patients (91%), with bilateral pneumonia seen in 32 of them (71%); the
percentage of patients with bilateral pneumonia was similar across dose cohorts. Viral load was
similar across the three cohorts, with average baseline values for SARS-CoV-2 RNA from
45
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprintthis version posted May 25, 2021. ; https://doi.org/10.1101/2021.05.25.21257505doi: medRxiv preprint